Since ribosomal RNA (rRNA) constitutes the majority of a total RNA sample, we recommend removing it to enrich the sample for other RNA types. This can be achieved through either a poly-A selection or rRNA depletion method, both of which help to focus the analysis on more informative RNA populations
RNA sequencing (RNA-seq) is a powerful tool for transcriptome analysis, and choosing between polyA enrichment and rRNA depletion depends on the specific goals of the experiment and the types of RNA you wish to study. Here’s a breakdown of when each method makes sense:
1. PolyA Enrichment
PolyA enrichment selectively isolates mRNA by binding to the polyA tails found in the 3’ end of most eukaryotic mRNAs. This is useful when you are primarily interested in coding RNA (mRNA), and it simplifies the analysis by reducing the complexity of the sample.
When to use PolyA Enrichment?
- Focus on mRNA: If your goal is to analyze protein-coding genes, polyA enrichment is ideal because it captures most of the mRNA that has a polyA tail (which is the majority of the transcriptome in eukaryotic cells).
- Eukaryotic Samples: Most eukaryotic mRNAs have polyA tails, making polyA enrichment particularly useful for these species.
- Reducing rRNA Interference: In RNA samples with high rRNA content, polyA enrichment helps reduce rRNA contamination by selectively enriching for mRNA, although it won’t remove rRNA entirely.
- mRNA Profiling: If you are performing transcriptomics to understand gene expression levels or alternative splicing in coding regions, polyA enrichment is effective.
- Limitations:
- Non-Polyadenylated RNAs: It won’t capture non-polyadenylated RNA, such as long non-coding RNAs (lncRNAs), miRNAs, rRNAs, and some small RNAs.
Considerations for Low-Quality RNA (PolyA Enrichment):
- Reduced mRNA Capture: If the mRNA is fragmented or degraded, polyA enrichment capture primarily the 3´end of mRNA, and there could be a loss of important data. This is often the case for RNA extracted from FFPE slides.
- Moderate Degradation: PolyA enrichment can still be useful when the RNA degradation is moderate (i.e., not severely fragmented), as long as there is a sufficient amount of intact polyadenylated RNA.
2. rRNA Depletion
rRNA depletion removes the vast majority of ribosomal RNA (rRNA), which makes up 80-90% of the total RNA in most eukaryotic cells. By depleting rRNA, you can analyze other types of RNA that may be less abundant, such as mRNA (in cases where polyA enrichment is not preferred), lncRNAs, small RNAs, and other non-coding RNAs.
When to use rRNA Depletion?
- Comprehensive Transcriptome Analysis: If your goal is to study all types of RNA, including non-coding RNAs (like lncRNAs and miRNAs), small RNAs, or even non-polyadenylated mRNAs, rRNA depletion is a better option because it preserves these RNA types.
- Prokaryotic Samples: In prokaryotes (which have rRNA as the most abundant RNA), rRNA depletion is commonly used to improve the quality of the RNA-seq data, as prokaryotic mRNAs do not have polyA tails.
- Limitations:
- Partial rRNA Removal: In some cases, rRNA depletion methods may not remove all rRNA species, leaving some residual.
Considerations for Low-Quality RNA (rRNA Depletion)
- Severe RNA Degradation: rRNA depletion is better suited for low-quality or degraded RNA, e.g. RNA extracted from FFPE slides, because it doesn’t rely on the integrity of polyA tails. Even with fragmented RNA, rRNA depletion can effectively reduce rRNA contamination.
Summary:
- PolyA Enrichment is optimal when you are focusing on mRNA and the RNA quality is high to moderate. It is less effective when RNA is degraded or when you need to analyze non-polyadenylated RNAs.
- rRNA Depletion works well for all non rRNA types and prokaryotic mRNA, especially if you want to capture a broader spectrum of RNA, including non-polyadenylated RNAs like lncRNAs. This method is also ideal when working with severely degraded RNA.